Use of proteasome inhibitor in the treatment of fibrotic diseases

ABSTRACT

The invention relates to the use of at least one proteasome inhibitor, preferably in the form of a threonine protease inhibitor, in the treatment of fibrotic diseases, espeially fibrotic diseases of the cardiovascular system.

The present invention relates to the use of at least one proteasomeinhibitor for the treatment of fibrotic diseases, especially fibroticdiseases of the cardiovascular system.

Myocardial fibrosis is a reaction to the overload of the myocardium,which may e.g. been caused by high blood pressure, myocardialinfarction, or cardiomyopathies. Numerous pathophysiological mechanismscontribute to this phenomenon, like e.g. the proliferation of heartconnective tissue cells leading to an increased matrix formation.Functional final states are the mass gain of the myocardium and anincreased fibrous reorganisation (fibrosis). One indication of theheart's reorganisation is thus the interstitial fibrosis leading to anincreased stiffness of the cardiac walls. This in respect to functionleads to a diastolic dysfunction, which may further aggravate theweakness of the myocardium. In part, these effects are caused by anincreased expression of genes coding for extracellular matrix proteinslike collagen I and III, the dominant collagens of the myocardium.

The currently best established strategy for the reduction of cardiacfibrosis is the pharmacological inhibition of the renin-angiotensinsystem, e.g. by means of ACE inhibitors or angiotensin II blockers.These substances however have the disadvantage that they will lead tointolerance effects in the patients in at least 5% of the cases.Moreover, they only affect the cardiac fibrosis mediated by therenin-angiotensin-system, but not the development of a myocardialfibrosis mediated by other agonists such as TGF-beta (transforminggrowth factor beta) or endothelin-1. There is thus the need foralternative methods, which could reduce or prevent a cardiac fibrosis.

It was now surprisingly found, that the inhibition of the proteasomesystem can effectively reduce or prevent a cardiac fibrosis, thusleading to improved cardiac function.

The subject of the present invention thus is the use of at least oneproteasome inhibitor for the treatment of fibrotic diseases. Thefibrotic diseases can affect very different organ systems like lung,liver, skin, joints, skeleton and/or glands. In particular, theinvention relates to fibrotic diseases of the cardiovascular system.

The ubiquitin-proteasome system is the main metabolic pathway for thedecomposition of intracellular proteins in eukaryotic cells, like e.g.signal mediators, cell cycle proteins and transcription factors. It wasshown, that an inhibition of the proteasome blocks cellularproliferation, interferes with different signal pathways and affectsgene expression (Lee, D. H. et al. (1998) Trends Cell Biol., 8397-403;Yu C.-L. et al. (1997) J. Biol. Chem., 272, 14017-14020; Heldin C.-H. etal. (1999) Nat. Cell Biol., 1, E195-E197 and Desterro, J. M. P. et al.,(2000) Cell Mol Live Sci., 57, 1207-1219).

Suitable as proteasome inhibitors are low-molecular organic compounds onthe one hand and molecular-biological compounds on the other hand,wherein these substances preferably inhibit the proteasome in asubstantially specific manner. The inhibition test can e.g. be performedas being described by Dahlmann B. et al. (2000) J. Mol. Biol., 303,643-653. The wording “substantially specific” according to the presentinvention means, that the inhibitor inhibits the proteasome in a morepronounced manner than other intracellular proteases like e.g. calpain,cathepsins or TPPII, wherein this inhibition is preferably about 10times stronger, in particular is about 100 times stronger, principallyis about 1000 times stronger than the inhibition of other intracellularproteases.

The inhibition values are commonly indicated as IC₅₀ (inhibitorconcentration at 50% inhibition) and compared with each other.Low-molecular organic compounds according to the present invention referto organic compounds with a relative molar mass ≦1000, preferably ≦800.Molecular-biological compounds according to the present invention referto nucleic acids, in particular to RNA or DNA, which inhibit theexpression of a component of the proteasomal system, e.g. thetranscription or translation of the proteasome encoding nucleic acids,or to proteins, in particular binding peptides or binding proteins,these substances being directed against at least one component of theproteasomal system, preferably against ubiquitin and/or against theproteasome. Said nucleic acid e.g. is an anti-sense-RNA or adouble-stranded RNA (dsRNA) against a proteasome encoding sequence, aduplex forming oligonucleotide against a proteasome encoding sequenceand/or a knock-out construct against a proteasome encoding sequence.Suitable as binding proteins or binding peptides e.g. are antibodies ortheir binding-reactive parts, e.g. single chain antibodies (scAb) orFab-fragments or derivatives thereof, e.g. bi-specific antibodiesagainst at least one component of the proteasomal system. A descriptionof the proteasomal system and of suitable proteasome inhibitors is e.g.found in Kisselev A. F. & Goldberg A. L. (2001) Chemistry & Biology, 8,739-758.

Suitable examples for proteasome inhibitors thus in particular arethreonine protease inhibitors, serine protease inhibitors and/orcysteine protease inhibitors, especially a peptide aldehyde, a peptideboronate, a peptide vinyl sulfone, a peptide epoxyketone, alactacystine, a peptide alpha keto-aldehyde, an alpha-ketoamide, anindanone peptide, a polyalkylene aldehyde and/or a polyphenol, inparticular a cathechin-3-gallate, which can e.g. be extracted from greentea. Especially suitable as proteasome inhibitor are Z-Leu-Leu-Leu-al(MG132), Z-Ile-Glu(OtBu)-Ala-Leu-al (PSI), CEP1612,pyrazylcarbonyl-Phe-Leu-boronate (PS-341), dansyl-Phe-Leu-boronate(DFLB), morpholino-naphthylalanine-Leu-boronate (MG273),NIP-Leu₃-vinylsulfone (NLVS), Tyr-Leu₃-VS, NIP-Leu-Leu-Asn-VS,Ada-Tyr-Ahx₃-Leu₃-VS, Ada-Lys(Bio)-Ahx₃-Leu₃-VS,Ac(Me)-Ile-Ile-Thr-Leu-EX (epoxomicin), dihydroeponemycin, lactacystine,clasto-lactacystine-beta-lactone (omuralide), PS-519, Ac-Leu-Leu-Nle-al(ALLN), 3,4-dichloroisocoumarine (DCI), 4-(2-aminoethyl)-benzenesulfonylfluoride (Pefablock SC), TMC-95A, gliotoxin,(−)-epigallocatechin-3-gallate (EGCG), ritonavir, lovastatin,aclacinomicin A (Aclarubicin) and/or cyclosporin, which are alldescribed more closely in Kisselev A. F. & Goldberg A. L. (2001, supra)and in FIG. 3, and wherein Z is a benzyloxycarbonyl group, al is analdehyde group, VS is a vinyl sulfone group, NIP is a3-nitro-4-hydroxy-5-iodophenylacetate group, and Bio is a biotin group.Particularly preferred of all these classes of compounds are threonineprotease inhibitors and, from this, especially the compounds MG132,MG262, lactacystine and/or epoxomicine, above all MG132 and/or MG262.

According to the present invention, the effects the proteasomeinhibition exerts on the cardiac fibrosis are studied in spontaneouslyhypertensive rats (SH-rats). In a long-term treatment of 12 weeks withthe specific proteasome inhibitor MG132 at a daily dose of 1 mg per kgwe surprisingly found a significant inhibition of the cardiac fibrosisby approximately 40%, moreover showing, that the inhibitor was very welltolerated. The described effects resulted in an improved ventricularfunction in the MG132-treated animals. What was shown in vitro was aconcentration-dependent inhibition of the growth of cardiac fibroblastsand a specific, concentration-dependent down-regulation of collagen Iα2by approximately 75% and of collagen IIIα1 by approximately 90%.Although one would have expected the systemic treatment with proteasomeinhibitors as inhibitors of a crucial cellular system to producecytotoxicity, the described long-term treatment did not result inmeasurable side effects, but in contrast was well tolerated. Thus,proteasome inhibitors show a preferable suitability for the treatment offibrotic diseases, in particular those of the cardiovascular system.

The present invention thus also relates to the treatment of patientssuffering from a fibrotic disease, preferably a fibrosis of thecardiovascular system. Possible as forms of application are both thesystemic and the local application.

The following methods and results with the corresponding FIGS. 1 to 3are intended to more closely illustrate the invention in an exemplarymanner, without limiting the invention to this.

DESCRIPTION OF THE FIGURES

FIG. 1 describes the effects of a MG132-treatment of SH-rats on cardiacfibrosis, determined by quantitative morphometry of siriusred-stained,left-ventricular microscopic sections (FIG. 1A-1C) and on cardiacfunction (FIG. 1D-1F).

FIG. 1A shows a representative section through the heart of an untreatedcontrol-SH-rat, which shows an increased myocardial fibrosis. Thefibrotic tissue is exemplarily indicated by asterisks. The bar equals 20μm.

FIG. 1B shows a typical section through the heart of a MG132-treatedanimal, which shows a significantly reduced cardiac fibrosis underproteasome inhibition.

FIG. 1C shows the quantitative evaluation by means of computer-aidedimage analysis. The result here is a cardiac fibrosis being reduced byapproximately 40% under MG132-treatment. Mean value ±S.E.M. (standarderror of mean=standard deviation/root of n), n=7-10, *:p<0,05.

FIG. 1D shows the end-diastolic pressures (LVEDP) in the left ventricleof the rats with a remarkably reduced pressure level in theMG132-treated animals. Mean value ±S.E.M., n=6-10, *:p<0,05, **p<0,01.

FIG. 1E shows the maximal pressure increase rate (dp/dtmax), which issignificantly higher in the MG132-treated animals. Mean value ±S.E.M.,n=6-10, *:p<0,05, **:p<0,01.

FIG. 1F shows the maximal pressure drop rate (dp/dtmin), which isincreased by a factor of 2 under treatment with MG132. Mean value±S.E.M., n=6-10, *:p<0,05, **:p<0,01.

FIG. 2 shows the effects of proteasome inhibitors on the proliferationand collagen expression in primary cardiac fibroblasts of the rat.

FIG. 2A shows the dose-dependent inhibition of proliferation by MG132.The cells were stimulated with 0,1 μM and 1 μM MG132 or 0,1% DMSO andcounted daily. Mean value ±S.E.M., n=3.

FIG. 2B shows the quantification of the mRNA-amount of the collagensIα1, Iα2 and IIIα1 by means of “real-time” PCR-analysis. UnderMG132-treatment, one observes a dose-dependent and dramatic reduction ofthe expression of the collagens Iα2 and IIIα1. Mean value ±S.E.M., n=2.

FIGS. 3A and B show the chemical structure of different proteasomeinhibitors.

FIG. 4 shows a real-time-PCR-analysis of the expression of MMP2 and MMP9and of the collagens Iα1, Iα2 and IIIα1 both under basal conditions(FIG. 4C or FIG. 4D) and under co-stimulation with MG132 and IL-β (FIG.4A or FIG. 4B).

FIG. 5 shows zymographic experiments for the detection of active MMP2and MMP9 under basal conditions (FIG. 5A) or under co-stimulation withproteasome inhibitor and IL-β (FIG. 5B).

FIG. 6 shows a band shift-analysis for the detection of active NFκB innuclear extracts after stimulation with MG132 and/or with IL-β.

METHODS

Method 1: Preparation of Cardiac Fibroblasts

Cardiac fibroblasts were prepared from neonatal Wistar rats by platingthe non-myocyte fraction of neonatal hearts. The hearts were removedfrom neonatal rats being 2-3 days old. Ventricular tissue was treatedover night at 4° C. with 50 mg/ml trypsin in Hanks-Balanced Salt Saline(HBSS) (Ca²⁺- and Mg²⁺-free, Invitrogen™, Karlsruhe, Germany) and withcollagenase (0,5 mg per ml) in Leibowitz medium (L-15, Invitrogen™,Karlsruhe, Germany) for 45 minutes at 37° C. After washing, the cellswere pre-plated at 37° C. for one hour. The adherent cells predominantlywere cardiac fibroblasts, which were then further cultivated understandard conditions in standard M199-medium with 10% new-born calfserum, L-glutamine and penicillin-streptomycin (Invitrogen™, Karlsruhe,Germany). In the experiments, we used sub-cultivated fibroblasts of thepassages 3-7.

Method 2: Proliferation Test

MG132, ALLM and MG262 were purchased at Calbiochem® (San Diego, Calif.,USA) and provided as 10 mM DMSO stock solutions. Cardiac fibroblasts(5×10⁴ cells per ml) were inoculated in 24-well-plates. Adherent cellswere either stimulated with MG132 (0,1 and 1 μM) or DMSO (0,1%) inmedium with 10% serum and further cultivated for 7 days. The medium wasexchanged every second day. The proliferation was determined by countingthe living cells in triplicate in daily intervals by means of thetrypanblue exclusion test. This method allows for the distinctionbetween living and dead cells: the dye trypanblue, which is added to thecells, can only enter into cells having a defective cell membrane, whichare consequently stained blue. Living cells with an intact cell membraneare not stained.

Method 3: Histological Determination of Fibrosis

For the pathohistological evaluation, the rat hearts were embedded intoparaffin, cut into sections having a thickness of 3 μm, subjected to ahaematoxylin-eosin and siriusred staining and analysed as beingdescribed in Hocher, B. et al., (1999), Hypertension, 3, 816-822. Thedegree of heart fibrosis was evaluated by means of quantitativemorphometry of the siriusred-stained sections with an image analysissystem (Quantimed 500, Image 1.61 program). In this analysis, the ratioof the red-stained tissue (connective tissue) and the total tissue ofthe section was determined.

Method 4: RNA and RT-PCR-Analysis

Total RNA was extracted by means of Trizol reagent (Invitrogen™,Karlsruhe, Germany). In the following, the RNA was subjected to reversetranscription (AMF Reverse Transkriptase, Roche Biochemicals, Mannheim,Germany) and treated with DNase (Ambion, Huntington, UK). We designedPCR-primers for the rat collagen-RNAs Iα1, Iα2, IIIα1, for the RNAs ofthe matrix-metalloproteinases (MMP) 2 and 9 and for the “housekeeping”gene hypoxanthine phosphoribosyltransferase (HPRT) by using thePrimerExpress-Software, version 1.2 (PerkinElmer/Applied Biosystems,Wellesley, Mass., USA); the respective primers were then obtained fromTIB Molbiol, Berlin, Germany. The PCR reaction was performed withSybrGreen in an ABI PRISM 5700 sequence detector (PerkinElmer/AppliedBiosystems, Wellesley, Mass., USA) according to the manufacturer'sinstructions. The relative quantification was performed by means to thecomparative CT-method as being described by the manufacturer.

Method 5: Animal Studies

Male SH-rats of the same age were obtained from the Jackson Laboratory,Maine, USA. The animals were maintained according to the internationalguidelines for the keeping und use of laboratory animals. 6-week-oldrats were treated for 12 weeks with MG132 (1 mg per kg body weight),0,1% DMSO as solvent control or with physiological saline (n=10 pergroup, daily intraperitoneal injection). The animals were fed with ahigh salt diet (drinking water with 2% sodium chloride). The bloodpressure was measured weekly by means of the standard tail-cuff method.The blood values and the standard laboratory markers of the renal andhepatic functions were determined 6 weeks after the treatment and at theend of the study by means of standard methods. At the end of the study,the animals were anaesthetised by means of an intraperitoneal injectionof a 20% urethane solution (0,9 g per kg body weight) and theleft-ventricular pressure parameters were determined as being describedby Saragoca, M. et al. (1981) Hypertension, 3, 380-385. The organs werewithdrawn, weighed and either shock-frozen in liquid nitrogen orembedded in paraffin for the histological analysis. Since no differencewas observed between the DMSO-treated and the salt-treated SH-rats inrespect to the above mentioned parameters, the DMSO-treated animalsserved as a control for the MG132-treatment.

The data was determined in the form of mean values ±S.E.M., if nototherwise indicated. The significance of the differences in theleft-ventricular pressure parameters and in the quantification of theheart fibrosis was determined according to Student's T-test. For thecell proliferation test, the significance was determined by comparingthe regression coefficients of MG132 in relation to the control group.An error probability of p<0,05 was considered as being significant. Thesoftware SPSS 9.0 was used for all statistical calculations.

Results

The treatment of spontaneously hypertensive rats with the specificproteasome inhibitor MG132 for 12 weeks was in general well toleratedduring this long period. In blood samples taken after 6 weeks and at theend of the study, neither changes in the differential blood picture nora change in the laboratory markers, which indicate side effects in thekidney or liver, were observed. Moreover, the MG132-treated animalsshowed no significant change in the systolic blood pressure and in theheart weight (table 1). TABLE 1 Control 1 mg/kg MG132 Systolic BD (mmHg) 196.75 ± 9.1   191.43 ± 11.2  Body Weight (g)  286 ± 36.6  280 ±15.3 Heart Weight (g) 1.28 ± 0.07 1.18 ± 0.07 HG/KG (mg/g) 4.64 ± 0.61 4.1 ± 0.34BD means blood pressure; KG means body weight and HG means heart weight.

As it can be seen in FIG. 1A to 1C, the MG132-treated SH-rats showed asignificant reduction of the heart fibrosis (−38%) in comparison to thecontrol animals, when determining the results by means of quantitativemorphometry of siriusred-stained sections of the left ventricles.

The prevention of cardiac fibrosis in MG132-treated SH-rats correlatedwell with the normal left-ventricular function, whereas the controlsshowed indications of a beginning left-ventricular dysfunction as shownin the FIGS. 1D-1F: The filling pressures (LVEDP) were significantlylower (FIG. 1D: 15±2 versus 5±3 mm Hg, p=0,017), the maximal pressureincrease rate dp/dtmax (FIG. 1E: 8010±538 versus 3375±662 mm Hg,p=0,003) and the maximal pressure drop rate dp/dtmin (FIG. 1F: −5046±726versus −2290±422 mm Hg/s, p=0,015)—which are parameters of cardiacinotropy and cardiac lusitropy—were more than twice higher in theMG132-treated SH-rats in comparison to the control SH-rats.

In order to elucidate possible mechanisms, by which the proteasomeinhibition may contribute in vivo to the reduced cardiac fibrosis,primary cardiac fibroblasts of the rat were treated with proteasomeinhibitors. As illustrated in FIG. 2A, the treatment of cardiacfibroblasts with 0,1 and 1 μM MG132 induced a dose-dependent inhibitionof proliferation. Moreover, the RNA-expression of collagen Iα2 and IIIα1was inhibited by MG132 in a dose-dependent manner by up to 73% and up to91% (FIG. 2B). In contrast to this, the expression of collagen Ial wasunaffected. A second specific proteasome inhibitor, MG262, which is aboronate derivative of MG132, inhibited the collagen expression in acomparably effective manner (FIG. 2B) and thus proves the specificity ofthe proteasomal inhibition. The cathepsin inhibitor ALLM, which as apeptide aldehyde (ALLM-al) is structurally related to MG132, in contrastshowed no effects on collagen expression (FIG. 2B).

The endemically occurring and—in respects of health policy—extremelyimportant organ fibroses (especially myocardial fibrosis) have to becompletely distinguished from extreme variants of fibrotic processes inthe form of inflammatory responses to foreign matters (see also capsuleformation in case of silicone implants), which are reported about by H.Rupp, P. Newrzella, H. König and B. Maisch in: “Hemmung der kardialenFibrose durch Blockierung des Transkriptionsfaktors NF-kappaB” at the67^(th) annual meeting of the “Deutsche Gesellschaft fürKardiologie-Herz-und Kreislaufforschung, Apr. 19-21, 2001, Mannheim.

Especially in the case of the myocardial fibrosis which occurs millionfold and is—according to the invention—treatable by the application ofproteasome inhibitors for prevention or therapy, the pathophysiologicalagent is the neuroendocrinological activation with the release ofvasoconstricting mediators like cathecholamins, angiotensin II,endothelin-1, TGF-beta and other factors, which are released in anincreased manner under the conditions of a systemic overload (e.g. achronic pressure stress in case of arterial hypertension) or a regionaloverload (e.g. compensatory hyperkinesis of the intact residualmyocardium in case of a myocardial infarction). Besides theirvasoconstricting properties, these mediators have strong growth inducingeffects in the sense of a mass gain of heart muscle cells and theproliferation and increased synthesis output in the form ofextracellular matrix formation of cardiac fibroblasts.

In case of fibrotic hearts damaged by overload and, according to theinvention, treated with proteasome inhibitors, normally no indicationfor an inflammatory reaction is observed in the exact tissue analysis.It is scientifically well proved that said cardiac fibroses beinginduced by overload are independent from the activation of theinflammatory transcription factor NFκB and follow the signal pathway ofTGF-beta (see Kitamoto et al., Circulation 2000; 102:806-12; Lijnen etal., Mol Gen Metab 2000; 71:418-35). The fibrosis of cardiovascularrelevance which according to the invention is treatable by proteasomeinhibitors is thus caused independently from NFκB by different forms ofoverload with consecutive neuroendocrinological activation.

These positions are clearly supported thereby, that the main substancegroups being directed against the development of a fibrosis orcontributing to the regression of a fibrosis have antagonistic effectson specific vasoactive mediators. Good examples for this are angiotensinconverting enzyme inhibitors (ACE inhibitors), angiotensin II type1-receptor antagonists (AT-1 antagonists) and endothelin receptorantagonists.

Other preferred forms of organ fibroses without a dominatinginflammatory component, which according to the invention are treatableby proteasome inhibitors are the liver fibrosis caused by congestion,the kidney fibrosis caused by high pressure and joint fibroses in caseof malpositions.

Experimental data of the present invention verify that the inventiveanti-fibrotic effects of proteasome inhibitors are independent from theactivation of the inflammatory transcription factor NFκB.

1. Real-Time PCR-Analysis of the Expression of Collagens and MMP2 and 9(FIG. 4):

For this analysis, cardiac fibroblasts were treated with proteasomeinhibitors for 24 hours under serum-free conditions. The prepared RNAwas then used to determine the expression of the collagens Iα1, Iα2 undIIIα1 and of MMP2 and MMP9 by means of real-time (RT)-PCR.

As it is shown in the FIGS. 4C and 4D, the inventive inhibition of theproteasome not only significantly inhibited the expression of thecollagens, but also the expression of the MMPs. This inventiveinhibitory effect of the proteasome inhibitors was observed under basalconditions and without an additional cytokine stimulation of the cells.

In order to investigate, if the proteasome inhibitors also show theseeffects after the induction of the MMPs caused by stimulation with theNFκB-activating cytokine interleukin-1β (IL-1β), the cardiac fibroblastswere co-stimulated with IL-1β and proteasome inhibitor.

The IL-1β-stimulation led to a significant increase of the expression ofthe MMPs, especially of MMP9 (FIG. 4A). This MMP has been described as aprotease, which can be activated by NFκB and stimulated by cytokinetreatment (Gum et al., J Biol. Chem. 1996; 271:10672-80). Thesimultaneous administration of proteasome inhibitors not only preventedthe IL-1β-induced increase of expression, but surprisingly reduced theexpression to less than the DMSO control values (FIG. 4A). Thisdemonstrates the inventive inhibition of the MMP-expression by means ofproteasome inhibition, which leads to an inhibition of theMMP-expression being stronger than the inhibition of the IL-1β mediatedMMP-expression, thus being a process independent from inhibiting theactivation of NFκB.

The inventive effect of the proteasome inhibition being independent fromNFκB-inhibition in the inventive prevention and treatment of fibroticdiseases was demonstrated even more impressively in an analysis ofcollagen expression under IL-1β stimulation (FIG. 4B).

As already being described by Siwik et al., (Siwik et al., Am J PhysiolCell Physiol. 2001; 280:C53-C60), the IL-1β treatment led to adown-regulation of the collagen expression in cardiac fibroblasts (FIG.4B). This suppression can be explained by down-regulating NFκB-elementsin the promoter of the collagens, i.e. by a suppressing effect ofactivated NFκB on collagen expression (Kouba et al., J. Immunol. 1999;162:3226-34). The co-stimulation of the fibroblasts with IL-1β and withproteasome inhibitors however did not result in a reversal of thissuppression of the collagens by NFκB as it would have been expected inan inhibition of the activation of NFκB by proteasome inhibitors, butsurprisingly led to an even stronger suppression of the collagenexpression than achieved with IL-1β alone (FIG. 4B).

The present investigations demonstrate that the inventive treatment offibrotic diseases by means of proteasome inhibitors is independent fromthe inhibition of the activation of NFκB. Advantageously, the inventionallows for the inhibition of the expression of MMPs and collagens infibrotic diseases in a manner being independent from the inhibition ofthe activation of NFκB. Preferably, the invention enables the preventionand therapy of fibrotic diseases, which are not or not predominantlymediated by NFκB.

2. Zymography for the Detection of Active MMP2 and MMP9 (FIG. 5):

The inventive inhibition of the expression of active MMP2 in cardiacfibroblasts under basal, i.e. not NFκB-stimulated conditions by means ofproteasome inhibitors was also verified in zymography experiments (FIG.5). In these experiments, cell culture supernatants were investigatedafter the incubation with proteasome inhibitors in the absence (FIG. 5A)and presence (FIG. 5B) of IL-1β in respect to their gelatinase activityby means of zymography.

According to the invention, the MMP2-formation is already reduced by theproteasome inhibitor in the absence of Il-1β, i.e. in a manner beingindependent from NFκB (FIG. 5A). The IL-1β-induced MMP2- andMMP9-activation was also reduced by the simultaneous administration ofproteasome inhibitors (FIG. 5B).

3. Band Shift Analysis for the Detection of Active NFκB (FIG. 6):

The above presented results show that the inventive inhibitory effectsof the proteasome inhibitors on collagen- and MMP-expression take placein non-stimulated cardiac fibroblasts and are thus independent from theinhibition of NFκB activation. This was additionally confirmed in bandshift analyses with oligonucleotides comprising a NFκB-DNA binding site.For this test, nuclear extracts from cardiac fibroblasts were prepared,which were treated with 0,5 μM MG132 or, respectively, with DMSO(solvent control) in a time course in the presence or absence of IL-1β.

As shown in FIG. 6, cardiac fibroblasts have no active NFκB complexunder non-stimulated, basal conditions (see control bands). Under IL-1β,stimulation, an activation of NFκB takes place, represented as bandshift. The simultaneous treatment with 0,5 μM MG132 took not less than 6hours to lead to a small inhibitory effect on NFκB, which however is inno way sufficient to explain the surprisingly strong reduction of theexpression of MMP2 and MMP9 in case of the simultaneous application ofMG132 and IL-1β (see above, FIG. 4A). After 24 h, one observed adecrease of the IL-1β-induced activation of NFκB with and without MG132.The in vitro experiments thus show that the inventive inhibitory effectsof proteasome inhibitors on the expression of collagens and MMPs aremediated independently from the inflammatory transcription factor NFκB.

Also the data presented at the beginning for the inventive prevention ofcardiac fibrosis in the animal model of the spontaneously hypertensiverats give evidence for the inventive, MG132-mediated suppression of acollagen expression being independent of inflammatory stimuli. Thisanimal model, according to the invention, does not see an inflammatorystimulus as the trigger for cardiac fibrosis, but a pronouncedhypertonia of the rats.

According to the invention, proteasome inhibitors do not only interferein an inhibitory manner with collagen- and MMP-expression, but displaytheir broad spectrum of activity also in respect to the suppression offibroblast proliferation.

The inventive therapeutic interventions preferably comprise the systemicapplication of proteasome inhibitors. In this approach, preferably atleast one proteasome inhibitor is administered to the patient in a dose,in which systemic side effects, in particular cytotoxic side effects, ofproteasome inhibitors are advantageously avoided or occur only to asmall degree. The inventive use of proteasome inhibitors for theprevention and therapy of fibroses is thus well tolerated, preferablyallows for a specific anti-fibrotic treatment and therefore enables aninventive prevention and therapy in a variety of patients.

In preferred embodiments, an inventive systemic application of at leastone proteasome inhibitor enables the prevention and treatment preferablyof a cardiac fibrosis, preferably of a cardiac fibrosis caused byoverload, preferably of a cardiac fibrosis caused by overload underchronic pressure stress in arterial hypertension, preferably of acardiac fibrosis caused by overload in compensatory hyperkinesia of theintact residual myocardium in case of myocardial infarction, preferablyof a cardiac fibrosis caused by overload with consecutiveneuroendocrinological activation, preferably of a cardiac fibrosis, incase of which a treatment with ACE inhibitors, AT-1-antagonists and/orendothelin receptor antagonists is indicated, preferably of a liverfibrosis caused by congestion, preferably of a kidney fibrosis caused byhigh pressure and preferably of all joint fibroses in case of jointmalpositions.

In the prevention and therapy of fibroses according to the invention, apatient is given at least one proteasome inhibitor, preferably in a doseof approximately 0,5 μg/kg body weight to approximately 0,5 mg/kg bodyweight, preferably in a dose of approximately 1 μg/kg body weight toapproximately 0,1 mg/kg body weight, preferably in a dose ofapproximately 0,01 mg/kg body weight to approximately 0,1 mg/kg bodyweight. These doses refer to all of the proteasome inhibitors mentionedin this specification, especially to the threonine protease inhibitors,in particular to MG132 and MG262. Preferably, at least one of theproteasome inhibitors mentioned at the beginning of this specificationis administered, preferably MG132. For this aim, one preferably usesadvantageous pharmaceutical formulations, which are familiar to theexpert, preferably solid and liquid medicinal preparations, preferablysolutions for infusion, which preferably contain at least oneadvantageous pharmaceutical additive, which is known to the expert andwhich contributes to the shelf life and/or the reduction of side effectsof the medical composition.

1. Use of at least one proteasome inhibitor for the treatment offibrotic diseases, which are not caused by inflammatory responses toforeign matters.
 2. Use according to claim 1 for the treatment of acardiac fibrosis caused by overload, a liver fibrosis caused bycongestion, a kidney fibrosis caused by high pressure or a jointfibrosis in case of a joint malposition.
 3. Use according to claim 2 forthe treatment of a cardiac fibrosis caused by overload under chronicpressure stress in arterial hypertension and/or for the treatment of acardiac fibrosis caused by overload in compensatory hyperkinesia of theintact residual myocardium in case of myocardial infarction.
 4. Useaccording to claim 2 for the treatment of a cardiac fibrosis, in which atreatment with ACE inhibitors, AT-1-antagonists and/or endothelinreceptor antagonists is indicated.
 5. Use according to claim 1, whereina patient is administered at least one proteasome inhibitor in a dose ofapproximately 0,5 μg/kg body weight to approximately 0,5 mg/kg bodyweight, preferably in a dose of approximately 1 μg/kg body weight toapproximately 0,1 mg/kg body weight, preferably in a dose ofapproximately 0,01 mg/kg body weight to approximately 0,1 mg/kg bodyweight.
 6. Use according to claim 1, characterised in that the fibroticdiseases relate to fibrotic organ diseases, preferably of the lung,liver, skin, joints, skeleton and/or glands, in particular to diseasesof the cardiovascular system.
 7. Use according to claim 1, characterisedin that the proteasome inhibitor is a low-molecular organic compound ora molecular-biological compound.
 8. Use according to claim 7,characterised in that the proteasome inhibitor is a threonine proteaseinhibitor, a serine protease inhibitor, a cysteine protease inhibitor, agene expression inhibitor of the proteasomal system and/or a bindingprotein or binding peptide directed against at least one component ofthe proteasomal system, preferably against ubiquitin and/or against theproteasome.
 9. Use according to claim 7, characterised in that theproteasome inhibitor is a peptide aldehyde, a peptide boronate, apeptide vinyl sulfone, a peptide epoxyketone, a lactacystine, a peptidealpha keto-aldehyde, an alpha-ketoamide, an indanone peptide, apolyalkylene aldehyde, a polyphenol, in particular acathechin-3-gallate, a nucleic acid directed against at least onecomponent of the proteasomal system and/or an antibody orbinding-reactive part or derivative thereof, directed against at leastone component of the proteasomal system.
 10. Use according to claim 7,characterised in that the proteasome inhibitor is Z-Leu-Leu-Leu-al(MG132), Z-Ile-Glu(OtBu)-Ala-Leu-al (PSI), CEP1612,pyrazylcarbonyl-Phe-Leu-boronate (PS-341), dansyl-Phe-Leu-boronate(DFLB), morpholino-naphthylalanine-Leu-boronate (MG273),NIP-Leu₃-vinylsulfone (NLVS), Tyr-Leu₃-VS, NIP-Leu-Leu-Asn-VS,Ada-Tyr-Ahx₃-Leu₃-VS, Ada-Lys(Bio)-Ahx₃-Leu₃-VS,Ac(Me)-Ile-Ile-Thr-Leu-EX (epoxomicin), dihydroeponemycin, lactacystine,clasto-lactacystine-beta-lactone (omuralide), PS-519, Ac-Leu-Leu-Nle-al(ALLN), 3,4-dichloroisocoumarine (DCI), 4-(2-aminoethyl)-benzenesulfonylfluoride (Pefablock SC), TMC-95A, gliotoxin,(−)-epigallocatechin-3-gallate (EGCG), ritonavir, lovastatin,aclacinomicin A (Aclarubicin), cyclosporin, an anti-sense-RNA or adouble-stranded RNA (dsRNA) against a proteasome encoding sequence, atriplex forming oligonucleotide against a proteasome encoding sequenceand/or a knock-out construct against a proteasome encoding sequence,wherein Z is a benzyloxycarbonyl group, al is an aldehyde group, VS is avinyl sulfone group, NIP is a 3-nitro-4-hydroxy-5-iodophenylacetategroup, and Bio is a biotin group.